1. Field of the Invention
The present invention relates generally to immunological assay techniques for determining the presence of an analyte in serum and more particularly to a two site cross-reaction immunoassay sandwich testing method which has particular application in the qualitative and quantitative determination of the level of the creatine phospho-kinase MB Isoenzyme (CK-MB) in human serum.
2. Description of the Prior Art
Conventional immunometric techniques depend upon the immunochemical reaction between a tagged antibody and the analyte to be assayed. Such antibodies are raised to specifically react with the particular analyte and may be tagged in a radioactive, fluorescent, chemiluminescent, enzymatic or other manner. However, many antibodies have cross-reactions with materials other than the specific analyte to be assayed whereupon the results of simple antibody-analyte tests become unreliable. To overcome this problem, sandwich type assay techniques have been developed which utilize two antibodies to sandwich the analyte therebetween.
Conventional sandwich techniques utilize three types of assays. In the first type, the undesired cross-reaction is reduced based on the observation that the undesirable cross-reactant would not react, at the same time, with antibodies specifically raised in two different animal species. Both of the antibodies are therefore raised against the specific analyte of interest, however they differ in that they are raised in different animal species, such as human beings and guinea pigs. Utilizing this sandwich technique, the undesirable effect of the unwanted cross-reactions of the first antibody is reduced through the utilization of the second antibody, whereby an accurate test for the analyte of interest is obtained. This type of sandwich assay technique is used in the detection of hepatitis.
The second type of sandwich assay is used when the serum contains, as metabolic by-products, fragments of the analyte to be measured. Two antibodies used for this assay are raised specifically against the analyte of interest. However, one antibody reacts specifically with one binding site the analyte while the second antibody reacts specifically with another binding site of the analyte. This prior art immunometric sandwich technique thus utilizes antibodies that are specifically raised against the specific analyte to be measured. The unwanted cross-reactions of the specific antibody with a fragment of the analyte of interest have caused researchers to develop this two site immunoassay sandwich technique which minimizes the effects of the unwanted cross-reactions from a fragment of the analyte.
The third sandwich technique uses the same specific antibody twice to detect the analyte of interest. In this technique an antibody which is specific to the analyte of interest is immobilized on a solid-phase and reacted with the analyte. All other interfering substances are removed before the analyte is reacted once again with the same antibody except that the antibody is now tagged for assay purposes.
As will be seen from the description of the preferred embodiment hereinbelow, the present invention differs from the prior art in that it specifically utilizes the previously avoided cross-reactive capabilities of two different antibodies to create a specific sandwich assay technique.